Avipox recombinants expressing foot and mouth disease virus genes

ABSTRACT

The present invention relates to modified poxyiral vectors and to methods of making and using the same. In particular, the invention relates to recombinant avipox that expresses gene products of foot and mouth disease virus (FMDV), and to compositions or vaccines that elicit immune responses directed to FMDV gene products and which can confer protective immunity against infection by FMDV.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/110,480, filed Apr. 20, 2005, which claims priority to provisionalU.S. application Ser. No. 60/563,786 filed on Apr. 20, 2004.

This application makes reference to U.S. application Ser. No.10/327,481, filed on Dec. 20, 2002, which is a continuation ofInternational application No. PCT/FR01/02042, filed on Jun. 27, 2001,published on Jan. 3, 2002 as WO 02/00251, and claiming priority toFrench application No. 00/08437, filed on Jun. 29, 2000.

All of the foregoing applications, as well as all documents cited in theforegoing applications (“application documents”) and all documents citedor referenced in the application documents are incorporated herein byreference. Also, all documents cited in this application (“herein-citeddocuments”) and all documents cited or referenced in herein-citeddocuments are incorporated herein by reference. In addition, anymanufacturer's instructions or catalogues for any products cited ormentioned in each of the application documents or herein-cited documentsare incorporated by reference. Documents incorporated by reference intothis text or any teachings therein can be used in the practice of theinvention. Documents incorporated by reference into this text are notadmitted to be prior art.

FIELD OF THE INVENTION

The present invention relates to vectors, such as viruses, e.g.,modified viruses such as poxviruses, and to methods of making and usingthe same. In particular, the invention relates to recombinant avipoxvectors and viruses that express antigens of foot and mouth diseasevirus (FMDV), and to methods of making and using the same. The inventionfurther relates to methods of eliciting an immune response to FMDV in asubject.

BACKGROUND OF THE INVENTION

Foot-and-mouth disease (FMD) is one of the most virulent and contagiousdiseases affecting farm animals. This disease is endemic in numerouscountries in the world, especially in Africa, Asia and South America. Inaddition, epidemic outbreaks can occur periodically. The presence ofthis disease in a country may have very severe economic consequencesresulting from loss of productivity, loss of weight and milk productionin infected herds, and from trade embargoes imposed on these countries.The measures taken against this disease consist of strict application ofimport restrictions, hygiene controls and quarantine, slaughtering sickanimals and vaccination programs using inactivated vaccines, either as apreventive measure at the national or regional level, or periodicallywhen an epidemic outbreak occurs.

FMD is characterized by its short incubation period, its highlycontagious nature, the formation of ulcers in the mouth and on the feetand sometimes, the death of young animals. FMD affects a number ofanimal species, in particular cattle, pigs, sheep and goats. The agentresponsible for this disease is a ribonucleic acid (RNA) virus belongingto the Aphthovirus genus of the Picornaviridae family (Cooper et al.,Intervirology, 1978, 10, 165-180). At present, at least seven types offoot-and-mouth disease virus (FMDV) are known: the European types (A, Oand C), the African types (SAT1, SAT2 and SAT3) and an Asiatic type(Asia 1). Numerous sub-types have also been distinguished (Kleid et al.Science (1981), 214, 1125-1129).

FMDV is a naked icosahedral virus of about 25 nm in diameter, containinga single-stranded RNA molecule consisting of about 8500 nucleotides,with a positive polarity. This RNA molecule comprises a single openreading frame (ORF), encoding a single polyprotein containing, interalia, the capsid precursor also known as protein P1 or P88. The proteinP1 is myristylated at its amino-terminal end. During the maturationprocess, the protein P1 is cleaved by the protease 3C into threeproteins known as VP0, VP1 and VP3 (or 1AB, 1D and 1C respectively;Belsham G. J., Progress in Biophysics and Molecular Biology, 1993, 60,241-261). In the virion, the protein VP0 is then cleaved into twoproteins, VP4 and VP2 (or 1A and 1B respectively). The mechanism for theconversion of the proteins VP0 into VP 1 and VP3, and for the formationof mature virions is not known. The proteins VP1, VP2 and VP3 have amolecular weight of about 26,000 Da, while the protein VP4 is smaller atabout 8,000 Da.

The simple combination of the capsid proteins forms the protomer or 5Smolecule, which is the elementary constituent of the FMDV capsid. Thisprotomer is then complexed into a pentamer to form the 12S molecule. Thevirion results from the encapsidation of a genomic RNA molecule byassembly of twelve 12S pentamers, thus constituting the 146S particles.The viral capsid may also be formed without the presence of an RNAmolecule inside it (hereinafter “empty capsid”). The empty capsid isalso designated as particle 70S. The formation of empty capsids mayoccur naturally during viral replication or may be produced artificiallyby chemical treatment.

Many hypotheses, research routes, and proposals have been developed inan attempt to design effective vaccines against FMD. Currently, the onlyvaccines on the market comprise inactivated virus. Concerns about safetyof the FMDV vaccine exist, as outbreaks of FMD in Europe have beenassociated with shortcomings in vaccine manufacture (King, A. M. Q. etal, (1981) Nature 293: 479-480). The inactivated vaccines do not conferlong-term immunity, thus requiring booster injections given every year,or more often in the event of epidemic outbreaks. In addition, there arerisks linked to incomplete inactivation and/or to the escape of virusduring the production of inactivated vaccines (King, A. M. Q., ibid). Agoal in the art has been to construct conformationally correctimmunogens lacking the infective FMDV genome to make effective and safevaccines.

Vaccinia virus has been used successfully to immunize against smallpox,culminating in the worldwide eradication of smallpox in 1980. Thus, anew role for poxviruses became important, that of a geneticallyengineered vector for the expression of foreign genes (Panicali andPaoletti, 1982; Paoletti et al., 1984). Genes encoding heterologousantigens have been expressed in vaccinia, often resulting in protectiveimmunity against challenge by the corresponding pathogen (reviewed inTartaglia et al., 1990). A highly attenuated strain of vaccines,designated MVA, has also been used as a vector for poxvirus-basedvaccines. Use of MVA is described in U.S. Pat. No. 5,185,146.

Additional vaccine vector systems involve the use of avipox viruses,which are naturally host-restricted poxviruses. Both fowlpoxvirus (FPV;Taylor et al. 1988a, b) and canarypoxvirus (CPV; Taylor et al., 1991 &1992) have been engineered to express foreign gene products. Fowlpoxvirus (FPV) is the prototypic virus of the Avipox genus of the Poxvirusfamily. The virus causes an economically important disease of poultrythat has been well controlled since the 1920's by the use of liveattenuated vaccines. Replication of the avipox viruses is limited toavian species (Matthews, 1982) and there are no reports in theliterature of avipox virus causing a productive infection in anynon-avian species including man. This host restriction provides aninherent safety barrier against transmission of the virus to otherspecies and makes the use of avipox virus based vaccine vectors inveterinary and human applications an attractive proposition.

Other attenuated poxvirus vectors have been prepared by geneticmodifications of wild type strains of virus. The NYVAC vector, derivedby deletion of specific virulence and host-range genes from theCopenhagen strain of vaccinia (Tartaglia et al., 1992) has proven usefulas a recombinant vector in eliciting a protective immune responseagainst an expressed foreign antigen. Another engineered poxvirus vectoris ALVAC, derived from canarypox virus (see U.S. Pat. No. 5,756,103).ALVAC does not productively replicate in non-avian hosts, acharacteristic thought to improve its safety profile (Taylor et al.,1991 & 1992). ALVAC was deposited under the terms of the Budapest Treatywith the American Type Culture Collection under accession numberVR-2547. Yet another engineered poxvirus vector is TROVAC, derived fromfowlpox virus (see U.S. Pat. No. 5,766,599).

Recombinant poxviruses can be constructed in two steps known in the artand analogous to the methods for creating synthetic recombinants ofpoxviruses such as the vaccinia virus and avipox virus described in U.S.Pat. Nos. 4,769,330; 4,722,848; 4,603,112; 5,110,587; 5,174,993;5,494,807; and 5,505,941, the disclosures of which are incorporatedherein by reference. It can thus be appreciated that provision of a FMDVrecombinant poxvirus, and of compositions and products therefrom,particularly ALVAC or TROVAC-based FMDV recombinants and compositionsand products therefrom, especially such recombinants containing the P1genes and/or C3 protease gene of FMDV, and compositions and productstherefrom, would be a highly desirable advance over the current state oftechnology.

SUMMARY OF THE INVENTION

Accordingly, one aspect of the present invention provides a recombinantavipox vector comprising at least one nucleic acid molecule encoding oneor more foot-and-mouth disease virus (FMDV) antigen(s). In advantageousembodiments, the avipox is ALVAC or TROVAC.

Advantageously, the FMDV antigen(s) can be VP1, VP2, VP3, VP4, 2A, 2B or3C. Advantageously, the nucleic acid molecule encoding one or morefoot-and-mouth disease virus (FMDV) antigen(s) is a cDNA encoding FMDVP1 region and a cDNA encoding FMDV 3C protease of FMDV.

In one embodiment, the FMDV antigens are operably linked to a promotersequence, which can be the H6 vaccinia promoter, I3L vaccinia promoter,42K vaccinia promoter, 7.5K vaccinia promoter, or Pi vaccinia promoter.In another embodiment, the promoter is the H6 vaccinia promoter, whichis mutated such that the expression levels of the FMDV antigens aredecreased compared with expression levels of the FMDV antigens under awild type (i.e. unmutated) H6 vaccinia promoter.

In another embodiment, the avipox vector of the present inventioncomprises a C6 insertion locus, wherein flanking sequences of the C6insertion locus promote homologous recombination of the FMDV antigenswith the C6 insertion locus. Advantageously, the flanking sequencescomprise the C6L and C6R open reading frames of canarypox.

In a further embodiment, the avipox vector of the present inventioncomprises a F8 insertion locus, wherein the flanking sequences of the F8insertion locus promote homologous recombination of the FMDV antigenswith the F8 insertion locus. Advantageously, the flanking sequencescomprise the F8L and F8R open reading frames of fowlpox.

A second aspect of the present invention provides a recombinant avipoxvirus, comprising at least one nucleic acid molecule encoding one ormore FMDV antigens. The present invention also provides recombinantavipox viruses vCP2186, vCP2181, vCP2176, and vFP2215.

A further aspect of the invention relates to a method of eliciting animmune response to FMDV in a subject, comprising administering theavipox vector or avipox virus of the present invention to the subject.

In yet another aspect of the present invention, a method of producing arecombinant avipox vector comprising at least one nucleic acid moleculeencoding one or more FMDV antigen(s), comprising the steps of: a)linearizing a donor plasmid with a restriction endonuclease, wherein thedonor plasmid comprises restriction endonuclease cleavage sites or amultiple cloning site; and b) ligating at least one nucleic acidmolecule comprising (i) a nucleic acid sequence encoding one or moreFMDV antigen(s), (ii) a viral promoter sequence, and (iii) insertionsequences flanking (i) and (ii) that have complementary restrictionendonuclease cleavage sites to the donor plasmid at FMDV antigens,thereby producing the recombinant avipox vector.

The method can further comprise the steps of c) introducing the vectorinto a cell permissive for replication of the vector; and d) isolatingthe vector from the cell. Advantageously, the cell permissive forreplication of the vector is a chicken embryonic fibroblast.

In another embodiment, the vector further comprises a reporter gene,which is selected from the group consisting of the neomycin resistancegene, the ampicillin resistance gene, lacZ (β-galactosidase),luciferase, and green fluorescent protein (GFP).

These and other objects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying drawings,incorporated herein by reference. Various preferred features andembodiments of the present invention will now be described by way ofnon-limiting examples and with reference to the accompanying drawings inwhich:

FIG. 1 shows the genome of foot and mouth disease virus (FMDV) and thegenes inserted into the avipox recombinants.

FIG. 2 shows the oligonucleotide primers used to PCR-amplify the H6pFMDV gene cassette (SEQ ID NO:1-3), and the amino acids encoded by thenucleotides (SEQ ID NO:4 and 5).

FIGS. 3A and 3B show the construction of a pC5 H6p FMDV P1+3C donorplasmid for generating ALVAC recombinants, with inserts at the C5 loci.

FIGS. 4A-4E show the nucleotide (SEQ ID NO:6) and amino acid sequences(SEQ ID NO:7) of the C5 H6p FMDV gene cassette of the pC5 H6p FMDV P1+3Cdonor plasmid.

FIGS. 5A and 5B show the construction of a pF8 H6p FMDV P1+3C donorplasmid for generating fowlpox recombinants, with the insert at theunique F8 locus.

FIGS. 6A-6F show the nucleotide (SEQ ID NO:8) and amino acid sequences(SEQ ID NO:9) of the F8 H6p FMDV gene cassette of the pF8 H6p FMDV P1+3Cdonor plasmid.

FIG. 7 shows the oligonucleotide primers used to PCR amplify the 3′-endof the FMDV gene cassette (SEQ ID NO:10-12), and the amino acids encodedby the nucleotides (SEQ ID NO:13 and 14).

FIG. 8 shows the construction of a promoter-less pC6 FMDV P1+3Cinsertion plasmid for introduction of different promoters.

FIGS. 9A and 9B show the construction of a pC6 H6p FMDV P1+3C donorplasmid for generating ALVAC recombinants, with the insert at the uniqueC6 locus.

FIGS. 1A-10E show the nucleotide (SEQ ID NO:15) and amino acid sequences(SEQ ID NO:16) of the C6 H6p FMDV gene cassette of the pC6 H6p FMDVP1+3C donor plasmid.

FIG. 11 shows the nucleotide sequences of the wild-type early/late H6promoter (H6p) (SEQ ID NO:17) and the mutant early H6 promoter (H6p*)(SEQ ID NO:18).

FIGS. 12A and 12B show the oligonucleotide primers used to amplify anH6p* 5′-FMDV fragment (SEQ ID NO:19-23) and the amino acids encoded bythe nucleotides (SEQ ID NO:24 and 25).

FIGS. 13A and 13B show the construction of a pC6 H6p* FMDV P1+3C donorplasmid for generating ALVAC recombinants, with the insert at the uniqueC6 locus.

FIGS. 14A-14E show the nucleotide (SEQ ID NO:26) and amino acidsequences (SEQ ID NO:27) of the C6 H6p* FMDV gene cassette of the pC6H6p* FMDV P1+3C donor plasmid.

FIGS. 15A and 15B show the oligonucleotide primers used to amplify theI3Lp 5′-FMDV fragment (SEQ ID NOS:28-33), and the amino acids encoded bythe nucleotides (SEQ ID NO:34 and 35).

FIGS. 16A and 16B show the construction of a pC6 I3Lp FMDV P1+3C donorplasmid for generating ALVAC recombinants, with the insert at the uniqueC6 locus.

FIGS. 17A-17E show the nucleotide (SEQ ID NO:36) and amino acidsequences (SEQ ID NO:37) of the C6 I3Lp FMDV gene cassettes of the pC6I3Lp FMDV P1+3C donor plasmid.

FIGS. 18A and 18B show the oligonucleotide primers used to amplify the42Kp 5′-FMDV fragment (SEQ ID NO:38-43) and the amino acids encoded bythe nucleotides (SEQ ID NO:44 and 45).

FIGS. 19A and 19B show the construction of a pC6 42Kp FMDV P1+3C donorplasmid for generating ALVAC recombinants, with the insert at the uniqueC6 locus.

FIGS. 20A-20E show the nucleotide (SEQ ID NO:46) and amino acidsequences (SEQ ID NO:47) of the C6 42Kp FMDV gene cassette of the pC642Kp FMDV P1+3C donor plasmid.

FIGS. 21A-21C show the oligonucleotide primers used to amplify andrepair the 7.5Kp 5′-FMDV fragment (SEQ ID NO:48-54), and the amino acidsencoded by the nucleotides (SEQ ID NO:55-57).

FIGS. 22A and 22B show the construction of a pC6 7.5K FMDV P1+3C donorplasmid for generating ALVAC recombinants, with the insert at the uniqueC6 locus.

FIGS. 23A-23E shows the nucleotide (SEQ ID NO:58) and amino acidsequences (SEQ ID NO:59) of the C6 7.5Kp FMDV gene cassette of the pC67.5Kp FMDV P1+3C donor plasmid.

FIGS. 24A-24E show the oligonucleotide primers used to amplify andrepair the Pip 5′-FMDV fragment (SEQ ID NO:60-77), and the amino acidsencoded by the nucleotides (SEQ ID NO:78-80).

FIGS. 25A and 25B show the construction of a pC6 Pip FMDV P1+3C donorplasmid for generating ALVAC recombinants, with the insert at the uniqueC6 locus.

FIGS. 26A-26E show the nucleotide (SEQ ID NO: 81) and amino acidsequences (SEQ ID NO:82) of the C6 Pip FMDV gene cassette of the pC6 PipFMDV P1+3C donor plasmid.

FIG. 27 describes the oligonucleotide primers used to PCR amplify anH6p* 5′-FMDV fragment for insertion into a pF8 donor plasmid (SEQ IDNO:83-86).

FIGS. 28A and 28B illustrate the construction of a pF8 H6p* FMDV P1+3Cdonor plasmid for generating fowlpox recombinants.

FIGS. 29A-29F depict the nucleotide (SEQ ID NO:87) and amino acid (SEQID NO:88) sequences of the F8 H6p* FMDV P1+3C gene cassette of the pF8H6p* FMDV P1+3C donor plasmid.

FIG. 30 shows the expression analysis of ALVAC recombinants containingthe FMDV P1+3C gene cassette under the I3L or 42K promoters.

DETAILED DESCRIPTION OF THE INVENTION

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

As used herein, the term “operably linked” means that the componentsdescribed are in a relationship permitting them to function in theirintended manner.

An “antigen” is a substance that is recognized by the immune system andinduces an immune response. A similar term used in this context is“immunogen”.

It is therefore an object of this invention to provide compositions andmethods for treatment and prophylaxis of infection with FMDV. It is alsoan object to provide a means to treat or prevent foot and mouth disease.

In one aspect, the present invention relates to a modified recombinantavipox vector expressing at least one nucleic acid sequences encodingfor one or more FMDV antigens. The viral vector according to the presentinvention is advantageously an avipox virus, such as fowlpox virus andcanarypox virus and more particularly, ALVAC or TROVAC. The modifiedrecombinant vector comprises a heterologous nucleic acid sequence, whichencodes an antigenic protein, e.g., derived from FMDV ORFs that areencoded by the P1 (comprising VP1, VP2, VP3, VP4, and 2A), 2B, and/or 3Cregions.

In another aspect, the present invention relates to a modifiedrecombinant avipox virus that includes, in a non-essential region of thevirus genome, at least one heterologous nucleic acid sequence thatencodes one or more antigens from FMDV, such as gene products of the P1gene (comprising VP1, VP2, VP3, VP4, 2A), 2B, and/or 3C.

In a still further aspect, the present invention relates to methods ofeliciting an immune response to FMDV in a subject, comprisingadministering the recombinant avipox vector of the present invention.The present invention also relates to methods of eliciting an immuneresponse to FMDV in a subject, comprising administering the recombinantavipox virus of the present invention. Advantageously, the avipox virusis selected from the group consisting of vCP2186, vCP2181, vCP2176, andvFP2215.

The virus used according to the present invention is advantageously apoxvirus, particularly an avipox virus, such as fowlpox virus orcanarypox virus. TROVAC refers to an attenuated fowlpox that was aplaque-cloned isolate derived from the FP-1 vaccine strain offowlpoxvirus that is licensed for vaccination of 1-day-old chicks. ALVACis an attenuated canarypox virus-based vector that was a plaque-clonedderivative of the licensed canarypox vaccine, Kanapox (Tartaglia et al.,1992). ALVAC-based recombinant viruses expressing extrinsic immunogenshave also been demonstrated efficacious as vaccine vectors (Tartaglia etal., 1993 a,b). This avipox vector is restricted to avian species forproductive replication. On human cell cultures, canarypox virusreplication is aborted early in the viral replication cycle prior toviral DNA synthesis. Nevertheless, when engineered to express extrinsicimmunogens, authentic expression and processing is observed in vitro inmammalian cells and inoculation into numerous mammalian species inducesantibody and cellular immune responses to the extrinsic immunogen andprovides protection against challenge with the cognate pathogen (Tayloret al., 1992; Taylor et al., 1991).

ALVAC and TROVAC have also been recognized as unique among avipoxvirusesin that the National Institutes of Health (“NIH”; U.S. Public HealthService), Recombinant DNA Advisory Committee, which issues guidelinesfor the physical containment of genetic material such as viruses andvectors, i.e., guidelines for safety procedures for the use of suchviruses and vectors, which are based upon the pathogenicity of theparticular virus or vector, granted a reduction in physical containmentlevel: from BSL2 to BSL1. No other avipoxvirus has a BSL1 physicalcontainment level. Even the Copenhagen strain of vaccinia virus—thecommon smallpox vaccine—has a higher physical containment level; namely,BSL2. Accordingly, the art has recognized that ALVAC and TROVAC have alower pathogenicity than other avipox viruses.

Advantageously, the avipox virus vector is an ALVAC or a canarypox virus(Rentschler vaccine strain), which was attenuated through 200 or moreserial passages on chick embryo fibroblasts, after which a master seedtherefrom was subjected to four successive plaque purifications underagar, from which a clone was amplified through five additional passages.The avipox virus vector can also be a fowlpox virus, or an attenuatedfowlpox virus such as TROVAC.

The invention further relates to the product of expression of theinventive recombinant avipox virus and uses therefor, such as to formantigenic, immunological or vaccine compositions for treatment,prevention, diagnosis or testing; and, to DNA from the recombinantavipox virus which are useful in constructing DNA probes and PCRprimers.

In one aspect, the present invention relates to recombinant avipoxviruses containing at least one nucleic acid sequence expressing one ormore antigens from FMDV, advantageously in a non-essential region of theavipox virus genome. The avipox virus can be a fowlpox virus, especiallyan attenuated fowlpox virus such as TROVAC, or a canarypox virus,especially an attenuated canarypox virus, such as ALVAC.

According to the present invention, the recombinant avipox virus andavipox viral vectors express at least one nucleic acid sequence encodingone or more FMDV antigens. In particular, any or all genes or openreading frames (ORFs) encoding FMDV antigens can be isolated,characterized and inserted into ALVAC recombinants. The resultingrecombinant avipox virus is used to infect an animal. Expression in theanimal of FMDV antigens results in an immune response in the animal toFMDV. Thus, the recombinant avipox virus of the present invention may beused in an immunological composition or vaccine to provide a means toinduce an immune response, which may, but need not be, protective. Themolecular biology techniques used are described by Sambrook et al.(1989).

The invention also contemplates FMDV antigens that can be delivered as anaked DNA plasmid or vector, or DNA vaccine or immunological orimmunogenic compositions comprising nucleic acid molecules encoding andexpressing in vivo an FMDV antigen(s).

The FMDV antigen of interest can be obtained from FMDV or can beobtained from in vitro and/or in vivo recombinant expression of FMDVgene(s) or portions thereof. The FMDV antigen of interest can also beprovided using synthetic FMDV sequences. The FMDV antigen of interestcan be, but are not limited to: L_(b), L_(ab), P1-2A (comprising VP1,VP2, VP3, VP4, and 2A); P2 (comprising 2B and 2C), and P3 (comprising3A, 3B, VPg, 3C, and 3D), or portions thereof. In an advantageousembodiment, the FMDV antigens are P1 and 3C. In a particularly preferredembodiment, the FMDV antigens are P1-2A or P1-2A, 2B. Reference is madeherein to U.S. patent application Ser. No. 10/327,481, relating toisolation of FMDV genome sequences, the contents of which areincorporated by reference.

Non-essential regions have been defined in the art (Johnson et al.,(1993) Virology 196: 381-401) for vaccinia virus. These sites, alsoreferred to herein as “insertion loci”, are described in U.S. Pat. Nos.6,340,462, and 5,756,103 for ALVAC, the contents of which areincorporated herein by reference, and include, but are not limited to,thymidine kinase (TK), hemagglutinin (HA), M2L, C6, and other loci. Inone embodiment, where canarypox is used, the insertion locus is C6. Inanother embodiment, where fowlpox is used, the insertion locus is F8.

Insertion of nucleic acid sequences encoding FMDV antigens can befacilitated by homologous recombination, wherein the FMDV sequence ofinterest is flanked by sequences corresponding to avipox viral openreading frames immediately adjacent to the insertion locus (hereinafterreferred to as “flanking sequences” or “insertion sequences”).Homologous recombination is facilitated by recognition of homologousflanking sequences, which promotes integration of the FMDV sequencesinto the insertion locus of interest. By way of example, insertion ofFMDV sequences into the C6 locus requires the presence of the C6L andC6R ORFs on either side of the nucleic acid sequence encoding the FMDVantigen of interest in the viral vector. Thus, advantageously theinsertion loci is C6 and the flanking sequences comprise C6L and C6R.Where the F8 insertion locus is used, the flanking sequences compriseF8L and F8R.

The recombinant viral vectors of the invention expressing FMDV antigenscan be replicated or produced in cells or cell lines, or in vivo in ahost or subject. One alternative embodiment consists of replicating thevector in cells permissive for replication of the vector.

It must be noted that avipox viruses can only productively replicate inor be passaged through avian species or avian cell lines such as, forexample, chicken embryonic fibroblasts or QT35. The recombinant avipoxviruses harvested from avian host cells, when inoculated into anon-avian vertebrate, such as a mammal, in a manner analogous to theinoculation of mammals by vaccinia virus, without productive replicationof the avipox virus. Despite the failure of the avipox virus toproductively replicate in such an inoculated non-avian vertebrate,sufficient expression of the virus occurs so that the inoculated animalresponds immunologically to the antigenic determinants of therecombinant avipox virus and also to the antigenic determinants encodedin exogenous genes therein. Thus, in an advantageous embodiment, whenavipox viruses or viral vectors are used, chicken embryonic fibroblastsor QT35 are preferred as the cells permissive for viral vectorreplication.

The recombinant viral vectors and recombinant viruses can containpromoters that are operably linked to the FMDV antigens of the presentinvention. The promoter is advantageously of poxyiral origin andadvantageously early or early-late promoters, which may be, inparticular, the promoter P11K of the vaccinia virus, I3L poxyiralpromoter, 42K poxyiral promoter, H6 poxyiral promoter, Pi poxyiralpromoter, P28K of the vaccinia virus, P160K ATI of the cowpox virus. Inparticular, the sequence driving the early transcription of anearly-late promoter can be used instead of the full-length promoter(Moss, B. (1990) Ann. Rev. Biochem. 59: 661-688; Mars, M. et al, (1987)J. Mol. Biol. 198: 619-631; Davison, A. et al (1989) J. Mol. Biol. 210:749-769; Vassef, A. (1987) Nucl. Acid. Res. 15: 1427-1443). The promoteris advantageously a weak promoter. The terms “strong promoter” and “weakpromoter” are known in the art and are defined by the relative frequencyof transcription initiation (times per minute) at the promoter.

The invention also provides for poxyiral promoters that are mutated. Thepresent inventors have found that expression of certain FMDV antigens isnot possible from strong poxyiral promoters. Without being bound bytheory, it is believed that high levels of expression of potentiallytoxic FMDV antigens can preclude formation of stable poxyiralrecombinants. Therefore, the present invention also comprehends the useof a mutated poxyiral promoter, such as, for example, a mutated H6promoter, such that the expression levels of the FMDV antigens aredecreased compared with expression levels of the FMDV antigens under awild type promoter (Davison, A. et al (1989) J. Mol. Biol. 210:749-769). The mutated H6 promoter of the instant invention can beconsidered a weak promoter.

The mutated H6 promoter taught herein contains a point mutation. Theinvention can also employ promoters other than H6, which contain pointmutations that reduce their frequency of transcription initiationcompared with the wild type promoter. In addition, other types ofmutated promoters are suitable for use in the instant invention. Forexample, U.S. application Ser. No. 10/679,520, incorporated herein byreference, describes a truncated form of the H6 promoter (see alsoDavison, A. et al (1989) J. Mol. Biol. 210: 749-769; Taylor J. et al.,Vaccine, 1988, 6, 504-508; Guo P. et al. J. Virol., 1989, 63, 4189-4198;Perkus M. et al., J. Virol., 1989, 63, 3829-3836).

The present invention also relates to a method of producing arecombinant avipox vector comprising FMDV antigens, comprising the stepsof linearizing a donor plasmid with a restriction endonuclease, whereinthe donor plasmid comprises restriction endonuclease cleavage sites or amultiple cloning site, and ligating at least one nucleic acid sequencecomprising (i) a nucleic acid sequence encoding one or more FMDVantigen(s), (ii) a viral promoter sequence, and (iii) insertionsequences flanking (i) and (ii) that have complementary restrictionendonuclease cleavage sites to the donor plasmid at FMDV antigens,thereby producing the recombinant avipox vector. Advantageously, themethod further comprises the steps of introducing the vector into a cellpermissive for replication of the vector, and isolating the vector fromthe cell.

By definition, a donor plasmid expression vector (or donor plasmid)includes a DNA transcription unit comprising a polynucleotide sequencecontaining the cDNA to be expressed and the elements necessary for itsexpression in vivo. The donor plasmid can also include a poxyiral earlytermination signal at the 3′ terminus of the foreign gene (Moss, B.(1990) Ann. Rev. Biochem. 59: 661-688). The circular, super-coiled oruncoiled plasmid form is preferred. The linear form also comes under thescope of this invention.

Methods for making and/or using vectors (or recombinants) for expressionand uses of expression products and products therefrom (such asantibodies) can be by or analogous to the methods disclosed in hereincited documents and documents referenced or cited in herein citeddocuments. See, for example, Sambrook et al. Molecular Cloning (1999).The invention also includes the use of the avipox vectors expressingFMDV antigens in the research setting. The recombinant avipox vectorsand recombinant avipox viruses can be used to transfect or infect cellsor cell lines of interest to study, for example, cellular responses toFMDV antigens, or signal transduction pathways mediated by FMDVantigens.

In the research setting, it is often advantageous to design recombinantvectors or viruses that comprise reporter genes that can be easilydetected by laboratory assays and techniques. Reporter genes are wellknown in the art and can comprise resistance genes to antibiotics suchas, but not limited to, ampicillin, neomycin, zeocin, kanamycin,bleomycin, hygromycin, chloramphenicol, among others. Reporter genes canalso comprise green fluorescent protein, the lacZ gene (which encodesβ-galactosidase), luciferase, and β-glucuronidase.

The invention also relates to a method of eliciting an immune responseagainst foot-and-mouth disease in a subject comprising administering therecombinant avipox vectors or recombinant avipox viruses according tothe present invention to the subject. The subject can be any animalwhich can become infected with FMDV, in particular, bovine, ovine,porcine or caprine species. Methods of administration and doses aredefined herein.

The recombinant avipox vectors and viruses expressing FMDV antigens oran expression product thereof, immunological, antigenic or vaccinecompositions or therapeutic compositions, can be administered via aparenteral route (intradermal, intramuscular or subcutaneous). Such anadministration enables a systemic immune response, or humoral orcell-mediated responses.

As used herein, the terms “immunogenic composition” and “immunologicalcomposition” and “immunogenic or immunological composition” cover anycomposition that elicits an immune response against the targeted FMDVantigen; for instance, after administration of injection into theanimal, elicits an immune response against the targeted FMDV antigen.The terms “vaccinal composition” and “vaccine” and “vaccine composition”covers any composition that induces a protective immune response againstthe FMDV antigen or which efficaciously protects against the antigenafter administration or injection into the animal. The invention alsocomprehends recombinant avipox viral vectors administered as a plasmidDNA vector or vaccine.

More generally, the inventive recombinant avipox viral vectors andrecombinant avipox viruses expressing FMDV antigens, antigenic,immunogenic, immunological or vaccine avipox virus-FMDV compositions ortherapeutic compositions, can be prepared in accordance with standardtechniques well known to those skilled in the pharmaceutical orveterinary arts. Such compositions can be administered in dosages and bytechniques well known to those skilled in the medical or veterinary artstaking into consideration such factors as the age, sex, weight, speciesand condition of the particular patient, and the route ofadministration.

The compositions can be administered alone, or can be co-administered orsequentially administered with compositions, e.g., with “other”immunological, antigenic or vaccine or therapeutic compositions therebyproviding multivalent or “cocktail” or combination compositions of theinvention and methods of employing them. Again, the ingredients andmanner (sequential or co-administration) of administration, as well asdosages can be determined taking into consideration such factors as theage, sex, weight, species and condition of the particular subject, andthe route of administration. In this regard, reference is made to U.S.Pat. No. 5,843,456, incorporated herein by reference, and directed torabies compositions and combination compositions and uses thereof.

Examples of compositions of the invention include liquid preparationsfor orifice, or mucosal, e.g., oral, nasal, anal, vaginal, peroral,intragastric, etc., administration such as suspensions, solutions,sprays, syrups or elixirs; and, preparations for parenteral,subcutaneous, intradermal, intramuscular or intravenous administration(e.g., injectable administration) such as sterile suspensions oremulsions. In such compositions, the recombinant avipox virus orrecombinant avipox viral vectors may be in admixture with a suitablecarrier, diluent, or excipient such as sterile water, physiologicalsaline, glucose or the like. The compositions can also be lyophilized.The compositions can contain auxiliary substances, such as wetting oremulsifying agents, pH buffering agents, adjuvants, gelling or viscosityenhancing additives, preservatives, flavoring agents, colors, and thelike, depending upon the route of administration and the preparationdesired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”,17th edition, 1985, incorporated herein by reference, may be consultedto prepare suitable preparations, without undue experimentation.

Compositions in forms for various administration routes are envisionedby the invention. And again, the effective dosage and route ofadministration are determined by known factors, such as age, sex,weight, condition and nature of the animal, as well as LD₅₀ and otherscreening procedures which are known and do not require undueexperimentation. Dosages of each active agent can be as in herein citeddocuments (or documents referenced or cited in herein cited documents)and/or can range from one or a few to a few hundred or thousandmicrograms, e.g., 1 μg to 1 mg, for an immunogenic, immunological orvaccine composition; and, 10⁴ to 10¹⁰ TCID₅₀ advantageously 10⁶ to 10³TCID₅₀ for an immunogenic, immunological or vaccine composition.

Recombinants or vectors can be administered in a suitable amount toobtain in vivo expression corresponding to the dosages described hereinand/or in herein cited documents. For instance, suitable ranges forviral suspensions can be determined empirically. The viral vector orrecombinant in the invention can be administered to an animal orinfected or transfected into cells in an amount of about at least 10³pfu; more advantageously about 10⁴ pfu to about 10¹⁰ pfu, e.g., about10⁵ pfu to about 10⁹ pfu, for instance about 10⁶ pfu to about 10⁸ pfu,with doses generally ranging from about 10⁶ to about 10¹⁰,advantageously about 10⁸ pfu/dose, and advantageously about 10⁷ pfu perdose of 2 ml. And, if more than one gene product is expressed by morethan one recombinant, each recombinant can be administered in theseamounts; or, each recombinant can be administered such that there is, incombination, a sum of recombinants comprising these amounts.

In vector or plasmid compositions employed in the invention, dosages canbe as described in documents cited herein or as described herein or asin documents referenced or cited in herein cited documents.Advantageously, the dosage should be a sufficient amount of plasmid toelicit a response analogous to compositions wherein the antigen(s) ofFMDV are directly present; or to have expression analogous to dosages insuch compositions; or to have expression analogous to expressionobtained in vivo by recombinant compositions. For instance, where DNAvaccines are administered, suitable quantities of each plasmid DNA inplasmid compositions can be 1 μg to 2 mg, advantageously 50 μg to 1 mg.Documents cited herein (or documents cited or referenced in herein citeddocuments) regarding DNA plasmid vectors may be consulted by the skilledartisan to ascertain other suitable dosages for DNA plasmid vectorcompositions of the invention, without undue experimentation.

However, the dosage of the composition(s), concentration of componentstherein and timing of administering the composition(s), which elicit asuitable immunological response, can be determined by methods such as byantibody titrations of sera, e.g., by ELISA and/or seroneutralizationassay analysis. Such determinations do not require undue experimentationfrom the knowledge of the skilled artisan, this disclosure and thedocuments cited herein. And, the time for sequential administrations canbe likewise ascertained with methods ascertainable from this disclosure,and the knowledge in the art, without undue experimentation.

The immunogenic or immunological compositions contemplated by theinvention can also contain an adjuvant. Particularly suitable adjuvantsfor use in the practice of the present invention are (1) polymers ofacrylic or methacrylic acid, maleic anhydride and alkenyl derivativepolymers, (2) immunostimulating sequences (ISS), such asoligodeoxyribonucleotide sequences having one or more non-methylated CpGunits (Klinman D. M. et al., Proc. Natl. Acad. Sci., USA, 1996, 93,2879-2883; WO98/16247), (3) an oil in water emulsion, such as the SPTemulsion described on p 147 of “Vaccine Design, The Subunit and AdjuvantApproach” published by M. Powell, M. Newman, Plenum Press 1995, and theemulsion MF59 described on p 183 of the same work, (4) cationic lipidscontaining a quaternary ammonium salt, (5) cytokines, (6) aluminumhydroxide or aluminum phosphate or (7) other adjuvants discussed in anydocument cited and incorporated by reference into the instantapplication, or (8) any combinations or mixtures thereof. The DNAvaccines or immunogenic or immunological compositions encompassed by theinvention can be formulated with a liposome, in the presence or absenceof an adjuvant as described above.

Other suitable adjuvants include FMLP(N-formyl-methionyl-leucyl-phenylalanine; U.S. Pat. No. 6,017,537)and/or acrylic acid or methacrylic acid polymer and/or a copolymer ofmaleic anhydride and of alkenyl derivative. The acrylic acid ormethacrylic acid polymers can be cross-linked, e.g., with polyalkenylethers of sugars or of polyalcohols. These compounds are known under theterm “carbomer” (Pharmeuropa, Vol. 8, No. 2, June 1996). A personskilled in the art may also refer to U.S. Pat. No. 2,909,462(incorporated by reference), which discusses such acrylic polymerscross-linked with a polyhydroxylated compound containing at least 3hydroxyl groups: in one embodiment, a polyhydroxylated compound containsnot more than 8 hydroxyl groups; in another embodiment, the hydrogenatoms of at least 3 hydroxyls are replaced with unsaturated aliphaticradicals containing at least 2 carbon atoms; in other embodiments,radicals contain from about 2 to about 4 carbon atoms, e.g., vinyls,allyls and other ethylenically unsaturated groups. The unsaturatedradicals can themselves contain other substituents, such as methyl. Theproducts sold under the name Carbopol® (Noveon Inc., Ohio, USA) areparticularly suitable for use as an adjuvant. They are cross-linked withan allyl sucrose or with allylpentaerythritol, as to which, mention ismade of the products Carbopol® 974P, 934P, and 971P.

As to the copolymers of maleic anhydride and of alkenyl derivative,mention is made of the EMA® products (Monsanto), which are copolymers ofmaleic anhydride and of ethylene, which may be linear or cross-linked,for example cross-linked with divinyl ether. Also, reference may be madeto J. Fields et al., Nature 186:778-780, 1960 (incorporated byreference).

With regard to structure, the acrylic or methacrylic acid polymers andEMA are advantageously formed by basic units having the followingformula:

in which:

-   -   R₁ and R₂, which can be the same or different, represent H or        CH₃    -   x=0 or 1, advantageously x=1    -   y=1 or 2, withx+y=2.

For EMA, x=0 and y=2 and for carbomers x=y=1.

These polymers are soluble in water or physiological salt solution (20g/l NaCl) and the pH can be adjusted to 7.3 to 7.4, e.g., by soda(NaOH), to provide the adjuvant solution in which the expressionvector(s) can be incorporated. The polymer concentration in the finalvaccine composition can range between 0.01 and 1.5% w/v, advantageously0.05 to 1% w/v and advantageously 0.1 to 0.4% w/v.

The cationic lipids containing a quaternary ammonium salt which areadvantageously but not exclusively suitable for plasmids, areadvantageously those having the following formula:

in which R₁ is a saturated or unsaturated straight-chain aliphaticradical having 12 to 18 carbon atoms, R₂ is another aliphatic radicalcontaining 2 or 3 carbon atoms and X is an amine or hydroxyl group.

Among these cationic lipids, preference is given to DMRIE(N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propaneammonium; WO96/34109), advantageously associated with a neutral lipid,advantageously DOPE (dioleoyl-phosphatidyl-ethanol amine; Behr J. P.,1994, Bioconjugate Chemistry, 5, 382-389), to form DMRIE-DOPE.

Advantageously, the plasmid mixture with the adjuvant is formedextemporaneously or contemporaneously with administration of thepreparation or shortly before administration of the preparation; forinstance, shortly before or prior to administration, theplasmid-adjuvant mixture is formed, advantageously so as to give enoughtime prior to administration for the mixture to form a complex, e.g.between about 10 and about 60 minutes prior to administration, such asapproximately 30 minutes prior to administration.

When DOPE is present, the DMRIE:DOPE molar ratio is advantageously about95:about 5 to about 5:about 95, more advantageously about 1:about 1,e.g., 1:1.

The DMRIE or DMRIE-DOPE adjuvant:plasmid weight ratio can be betweenabout 50:about 1 and about 1:about 10, such as about 10:about 1 andabout 1:about 5, and advantageously about 1:about 1 and about 1:about 2,e.g., 1:1 and 1:2.

A recombinant vaccine or immunogenic or immunological composition canalso be formulated in the form of an oil-in-water emulsion. Theoil-in-water emulsion can be based, for example, on light liquidparaffin oil (European Pharmacopea type); isoprenoid oil such assqualane, squalene, EICOSANE™ or tetratetracontane; oil resulting fromthe oligomerization of alkene(s), e.g., isobutene or decene; esters ofacids or of alcohols containing a linear alkyl group, such as plantoils, ethyl oleate, propylene glycol di(caprylate/caprate), glyceryltri(caprylate/caprate) or propylene glycol dioleate; esters of branchedfatty acids or alcohols, e.g., isostearic acid esters. The oiladvantageously is used in combination with emulsifiers to form theemulsion. The emulsifiers can be nonionic surfactants, such as esters ofsorbitan, mannide (e.g., anhydromannitol oleate), glycerol,polyglycerol, propylene glycol, and oleic, isostearic, ricinoleic, orhydroxystearic acid, which are optionally ethoxylated, andpolyoxypropylene-polyoxyethylene copolymer blocks, such as the Pluronic®products, e.g., L121. The adjuvant can be a mixture of emulsifier(s),micelle-forming agent, and oil such as that which is available under thename Provax® (IDEC Pharmaceuticals, San Diego, Calif.).

The term “prime-boost” refers to the successive administrations of twodifferent types of vaccine or immunogenic or immunological compositionshaving at least one antigen in common. The priming administration(priming) is the administration of a first vaccine or immunogenic orimmunological composition type and may comprise one, two or moreadministrations. The boost administration is the administration of asecond vaccine or immunogenic or immunological composition type and maycomprise one, two or more administrations, and, for instance, maycomprise or consist essentially of annual administrations.

Thus, the invention encompasses prime-boost immunization or vaccinationmethod of an animal against at least one FMDV antigen comprisingadministering to the animal a priming DNA vaccine or immunological orimmunogenic composition comprising nucleic acid molecule(s) encoding andexpressing in vivo an antigen(s) from FMDV, and thereafter administeringa boosting composition that comprises the FMDV antigen expressed by theDNA vaccine or immunogenic or immunological composition, or arecombinant or modified vector, e.g., virus, such as an avipox virus(such as ALVAC, canarypox, TROVAC, or fowlpox virus) that contains andexpresses in an animal host cell a nucleotide sequence encoding theantigen of FMDV expressed by the DNA vaccine or immunogenic orimmunological composition. The boosting vaccine or immunogenic orimmunological composition can be the same as or different than thepriming vaccine or immunogenic or immunological composition.

For instance, the boosting vaccine or immunogenic or immunologicalcomposition can be advantageously the FMDV antigen expressed by the DNAvaccine (or immunogenic or immunological composition) and/or arecombinant or modified avipox vector, e.g., virus, vaccine orimmunogenic or immunological composition. A recombinant or modifiedvector is advantageously an in vivo expression vector, such as amodified or recombinant bacteria, yeast, virus, e.g. avipox virus,comprising nucleic acid molecule(s) encoding and expressing in vivo theantigen(s) from FMDV expressed by the DNA vaccine or immunogenic orimmunological composition. The boost is advantageously performed with aninactivated vaccine or immunogenic or immunological composition, or witha vaccine or immunogenic or immunological composition comprising arecombinant live viral vector, such as a recombinant avipox virus, thatcomprises nucleic acid molecule(s) encoding and expressing in vivo theantigen(s) from the FMDV antigen expressed by the DNA vaccine orimmunogenic or immunological composition. Thus, it is advantageous thatthe boost either comprises the antigen expressed by the DNA vaccine orimmunogenic or immunological composition or expresses in vivo the sameFMDV antigen expressed by the DNA vaccine or immunogenic orimmunological composition. Advantageously, the boost comprises therecombinant avipox virus expressing FMDV antigens described herein.

Alternatively, the prime-boost immunization or vaccination method cancomprise administering to the animal a priming vaccine comprising therecombinant avipox viruses of the present invention, and boostingthereafter with the DNA vaccine.

The DNA plasmid, or recombinant avipox vector expressing one or morenucleic acid sequences encoding at least one FMDV antigen, e.g., vectoraccording to this disclosure, can be preserved and/or conserved andstored either in liquid form, at about 5° C., or in lyophilised orfreeze-dried form, in the presence of a stabilizer. Freeze-drying can beaccording to well-known standard freeze-drying procedures. Thepharmaceutically acceptable stabilizers may be SPGA (sucrose phosphateglutamate albumin; Bovarnik et al., J. Bacteriology 59:509, 1950),carbohydrates (e.g., sorbitol, mannitol, lactose, sucrose, glucose,dextran, trehalose), sodium glutamate (Tsvetkov T et al, Cryobiology20(3): 318-23, 1983; Israeli E et al., Cryobiology 30(5): 519-23, 1993),proteins such as peptone, albumin or casein, protein containing agentssuch as skimmed milk (Mills C K et al, Cryobiology 25(2): 148-52, 1988;Wolff E et al., Cryobiology 27(5):569-75, 1990), and buffers (e.g.,phosphate buffer, alkaline metal phosphate buffer). An adjuvant and/or avehicle or excipient may be used to make soluble the freeze-driedpreparations.

The invention will now be further described by way of the followingnon-limiting Examples, given by way of illustration.

EXAMPLES Example 1 Construction of a pC5 H6p FMDV P1+3C Donor Plasmidfor Introduction of FMDV Genes into the C5 Loci of ALVAC

Plasmid pAd5-A24 was used as the donor plasmid to generate theadenovirus Ad5A24 recombinant. It is a ˜39 kb plasmid containing thestrain A24 P1 genes and the strain A12 3C protease. Several deletions ofthe FMDV genome were made for safety reasons and are indicated in FIG.1.

Plasmid pAd5-A24 was digested with EcoRI and XbaT and the ˜3.4 kbfragment containing the FMDV genes was inserted in pUC8:2 (pUC8 withBglII and XbaI sites added to the multiple cloning site). The resulting6 kb pUC FMDV plasmid (designated pHM-1119-1) was used as the source ofthe FMDV genes in all future constructs.

The H6 promoter (H6p) is an early/late promoter derived from thevaccinia H6 gene (Perkus, M. E. et al, (1989) J. Virol. 63: 3829-3836),which is designated as the H5 gene in the Copenhagen vaccinia strain.The H6p is a strong promoter that has been used extensively in avipoxrecombinants for foreign gene expression.

Plasmid pHM-1119-1 was used as the template for PCR amplification withprimers 11277.SL and 11282.SL. These primers introduced the 3′ end ofthe vaccinia H6 promoter, as well as translation and transcription stopsignals, and XbaI or BamHI restriction sites for cloning. The primersequences are shown in FIG. 2. The 3.4 kb PCR product was cloned intopCR2.1 to generate plasmid pHM-1151-4, pCR2.1 H6p FMDV.

Plasmid pCXL-148-2 is an ALVAC insertion plasmid for the C5 loci, whichcontains the vaccinia virus H6 promoter. The 3.4 kb NruI-XbaI fragmentfrom pHM-1151-4 was inserted into pCXL-148-2, to generate pC5 H6p FMDVP1+3C (pHM-1175-1). The construction of pHM-1175-1 is illustrated inFIGS. 3A and 3B and the sequence of the C5 H6p FMDV gene cassette isshown in FIGS. 4A-4E.

Despite multiple attempts, no ALVAC recombinants were generated from pC5H6p FMDV P1+3C, pHM-1175-1.

Example 2 Construction of a pF8 H6p FMDV P1+3C Donor Plasmid forIntroduction of FMDV Genes into the F8 Locus of Fowlpox

Plasmid pSL-6427-2-1 (pF8 H6p) is a fowlpox insertion plasmid, whichcontains the vaccinia virus H6 promoter. The 3.4 kb NruI-BamHI fragmentfrom pHM-1151-4 (pCR2.1 H6p FMDV; see Example 1) was inserted intopSL-6427-2-1, generating vector pHM-1180-11 (pF8 H6p FMDV P1+3C). Theconstruction of pHM-1180-11 is illustrated in FIGS. 5A and 5B and thesequence of the F8 H6p FMDV gene cassette is shown in FIGS. 6A-6F.

Despite multiple attempts, no fowlpox recombinants could be generatedfrom pF8 H6p FMDV P1+3C, pHM-1180-1.

Example 3 Construction of a Promoter-Less pC6 FMDV P1+3C InsertionPlasmid

The failure to generate avipox recombinants expressing FMDV genes couldbe due to the use of the strong vaccinia virus H6 promoter in the pC5H6p FMDV P1+3C and pF8 H6p FMDV P1+3C plasmids described in Examples 1and 2. In addition, the ALVAC donor plasmid results in the insertion ofgene cassettes at the two C5 loci. For ALVAC, different viral promotersand the unique C6 insertion locus was used.

Plasmid pHM-1119-1 (pUC FMDV, see Example 1) was used as the templatefor PCR amplification of a 3′-fragment of FMDV, with primers 11280.SLand 11352.CXL. The ˜900 bp PCR fragment contains the 3′-end of FMDV fromthe XhoI site and introduces translational and transcriptional stops anda PstI cloning site. The primers are illustrated in FIG. 7. The PCRfragment was cloned into pCR2.1, generating plasmid pHM-1240-2, pCR2.13′-FMDV.

Plasmid pC6L is an ALVAC insertion plasmid for the unique ALVAC C6 site.The ˜2.6 kb EcoRI-Xho 5′-FMDV fragment from pHM-1119-1 was inserted intopC6L, generating plasmid pCXL-1008-1, pC6 5′-FMDV. The ˜900 bp XhoI-PstIfragment from pHM-1240-2 was inserted into pCXL-1008-1, generatingpCXL-1013-2, pC6 FMDV. The construction of pC6 FMDV is illustrated inFIG. 8.

Example 4 Construction of a pC6 H6p FMDV P1+3C Donor Plasmid forInsertion of the FMDV Gene Cassette at the Unique C6 Locus of ALVAC

Plasmid pSL-6407-7 is a pC6 H6p insertion plasmid for the ALVAC C6locus, which contains the vaccinia virus H6 promoter. The H6 promoter isin the opposite orientation to the C6 arms. The ˜2.6 kb NruI-Xho 5′-FMDVfragment from pHM-1151-4 (pCR2.1 FMDV, see Example 1) was inserted intopSL-6407-7, generating pC6 H6p 5′-FMDV, pCXL-1008-3. The ˜900 bpXho-PstI 3′-FMDV fragment from pHM-1240-2 (pCR2.1 3′-FMDV, see Example3) was inserted into pCXL-1008-3, generating pC6 H6p FMDV P1+3C,pCXL-1013-4. The construction of pC6 H6p FMDV P1+3C is illustrated inFIGS. 9A and 9B and the sequence of the C6 H6p FMDV gene cassette isshown in FIGS. 10A-10E.

Despite multiple attempts, ALVAC recombinants could not be generatedusing the pC6 H6p FMDV P1+3C donor plasmid, suggesting that insertion ata single site with a strong promoter was not feasible.

Example 5 Construction of a pC6 H6p* FMDV P1+3C Donor Plasmid forInsertion of the FMDV Gene Cassette at the Unique C6 Locus of ALVAC

Based upon studies with the vaccinia virus 7.5K early promoter (Davison,A. J. and Moss, B. (1989) J. Mol. Biol. 210: 749-769), a point mutationwas introduced into the vaccinia virus H6 early promoter region,generating a mutant H6 promoter, H6p*. The wild-type early/late H6p andmutant early H6p* sequences are shown in FIG. 11.

Plasmid pHM-1119-1 (pUC FMDV, see Example 1) was used as the template toPCR amplify the H6p* 5′-FMDV fragment, with primers 11353.CXL and11358.CXL. The ˜1.2 kb fragment contained the H6p* and the 5′-FMDV genesup to a unique NdeI site. The fragment was cloned into pCR2.1,generating plasmid pHM-1249-1-3. This clone was missing a nucleotide inVP4, so site-directed mutagenesis was performed with primers 11410.HMand 11411.HM to repair the PCR error. Clone pHM-1260-2, pCR2.1 H6p*5′-FMDV, was confirmed by sequence analysis. FIG. 12A describes the PCRamplification primers and FIG. 12B describes the mutagenesis primers.

The ˜1.2 kb EcoR I-Nde I H6p* 5′-FMDV fragment from pHM-1260-2 wasinserted into pCXL-1013-2 (pC6 FMDV P1+3C, see Example 3), generatingplasmid pHM-1273-1, pC6 H6p* FMDV P1+3C. The construction of pC6 H6p*FMDV P1+3C is illustrated in FIGS. 13A and 13B and the sequence of theC6 H6p* FMDV gene cassette is shown in FIGS. 14A-14E.

To generate an ALVAC recombinant, primary chicken embryonic fibroblasts(CEF) were transfected with SapI-linearized pHM-1273-1 donor plasmid, inthe presence of FuGENE-6® reagent (Roche). The transfected cells weresubsequently infected with ALVAC as rescue virus at an MOI of 10 andafter 24 hours, the transfected-infected cells were harvested,sonicated, and used for recombinant virus screening. Recombinant plaqueswere screened based on the plaque lift hybridization method using a 1.7kb FMDV-specific probe labeled with horseradish peroxidase (HRP)according to the manufacturer's protocol (Amersham). ALVAC recombinantswere generated and designated as vCP2176.

Example 6 Construction of a pC6 T3Lp FMDV P1+3C Donor Plasmid forIntroduction of the FMDV Genes into the Unique C6 Locus of ALVAC

The early/intermediate I3L promoter (I3Lp) from vaccinia virus (Schmitt,J. F. and Stunnenberg, H. G. (1988) J. Virol. 62: 1889-1897) has beenused previously in avipox recombinants.

Plasmid pCXL-1-4 is pC5 H6p EHV-1 gB (−TM)/42Kp EHV-1gD (−TM)/I3Lp EHV-1gC (−TM), a donor plasmid used to introduce the EHV-1 gB, gC, and gDgenes into ALVAC (described in U.S. Pat. No. 5,756,103). Each geneutilizes a different viral promoter, so pCXL-1-4 was used as thetemplate to PCR amplify the I3L promoter. Primers 11407.CXL and11423.CXL were used to amplify a 75 bp fragment containing the I3 Lpromoter and the 5′-end of the FMDV genes. The PCR primers are describedin FIG. 15A.

A 648 bp PCR fragment, which contains a 20 bp overlap with the 75 bpI3Lp fragment, was amplified using primers 11425.CXL and 11407.CXL, withpHM-1119-1 (pUC FMDV, see Example 1) as template. This fragmentcontained the 5′-FMDV genes up to the unique KpnI site. The PCR primersare described in FIG. 15B.

The two PCR fragments were mixed at a 1:1 molar ratio and PCR amplifiedusing primers 11423.CXL and 11407.CXL. The resultant 703 bp fragment wascloned into pCR2.1, generating pCXL-1068-1 (pCR2.1 I3Lp 5′-FMDV).

The ˜700 bp EcoRI-KpnI I3Lp 5′-FMDV fragment from pCXL-1068-1 wasinserted into pHM1119-1, generating pCXL-1072-2 (pUC I3Lp FMDV P1+3C).

The ˜1.2 kb EcoRI-NdeI I3Lp 5′-FMDV fragment from pCXL-1072-2 wasinserted into pCXL-1013-2 (pC6 FMDV P1+3C). The construction of pC6 I3LpFMDV P1+3C is illustrated in FIGS. 16A and 16B and the sequence of theC6 I3Lp FMDV gene cassette is shown in FIGS. 17A-17E.

To generate an ALVAC recombinant, primary CEFs were transfected with 20μg of SapI-linearized donor plasmid pCXL-1079-1 using FuGENE-6® reagent(Roche). The transfected cells were subsequently infected with ALVAC asrescue virus at an MOI of 10 and after 24 hours, thetransfected-infected cells were harvested, sonicated, and used forrecombinant virus screening. Recombinant plaques were screened based onthe plaque lift hybridization method using a 1.7 kb FMDV-specific probelabeled with horseradish peroxidase (HRP) according to themanufacturer's protocol (Amersham). After four sequential rounds ofplaque purification, the recombinants designated as vCP2181.4.1.1.1 andvCP2181.5.1.1.1 were generated and confirmed by hybridization as 100%positive for the FMDV insert and 100% negative for the C6 ORF.

Single plaques were selected from the 4^(th) round of plaquepurification and expanded to obtain P1 (1×T25 flask per sister), P2(1×T75 flask per sister) and P3 (4×roller bottles per sister) amplifiedstocks of the vCP2181 recombinants. The infected cells from the rollerbottles were harvested and concentrated to produce virus stock. Theviral concentrate was re-confirmed by hybridization of plaque lifts withthe FMDV- and C6-specific probes. Viral DNA was prepared and the correctinsertion of the FMDV gene cassette at the ALVAC C6 locus was confirmedby Southern blot and sequence analyses. Immunoblot and immunoplaqueassays were performed using specific antibodies as described in Example7 (see FIG. 30).

Example 7 Construction of a pC6 42 kDp FMDV P1+3C Donor Plasmid for theIntroduction of the FMDV Genes into the Unique C6 Locus of ALVAC

A 42K promoter (42Kp) derived from the AMV091 gene (vaccinia virus A23Rhomolog) of the insect poxvirus Amsacta moorei (Bawden, A. L. et al,(2000) Virology 274: 120-139) has been used previously in avipoxrecombinants (U.S. Pat. No. 5,756,103).

Plasmid pCXL-1-4 is pC5 H6p EHV-1 gB (−TM)/42Kp EHV-1 gD (−TM)/I3LpEHV-1 gC (−TM), a donor plasmid used to introduce the EHV-1 gB, gC, andgD genes into ALVAC (see Example 6). Each gene uses a different viralpromoter, so pCXL-1-4 was used as the template to PCR amplify the 42Kpromoter. Primers 11426.CXL and 11427.CXL were used to amplify a 48 bpfragment containing the 42K promoter and the 5′-end of the FMDV genes.The PCR primers are described in FIG. 18A.

A 647 bp PCR fragment, which contains a 20-bp overlap with the 48 bp42Kp fragment, was amplified using primers 11428.CXL and 11407.CXL, withpHM-1119-1 (pUC FMDV, see Example 1) as a template. This fragmentcontains the 5′-FMDV genes up to the unique KpnI site. The PCR primersare described in FIG. 18B.

The two PCR fragments were mixed at a 1:1 molar ratio and PCR amplifiedusing primers 11426.CXL and 11407.CXL. The resultant 676 bp fragment wascloned into pCR2.1, generating pCXL-1080-2-2 (pCR2.1 42Kp 5′-FMDV).

The 676 bp EcoRI-KpnI 42Kp 5′-FMDV fragment from pCXL-1080-2-2 wasinserted into pHM-1119-1, generating pCXL-1089-1 (pUC 42Kp FMDV P1+3C).

The ˜1.2 kb EcoRI-NdeI 42Kp 5′-FMDV fragment from pCXL-1089-1 wasinserted into pCXL-1013-2 (pC6 FMDV P1+3C, see Example 3), generatingpCXL-1095-1 (pC6 42Kp FMDV P1+3C). The construction of pC6 42Kp FMDVP1+3C is illustrated in FIGS. 19A and 19B and the sequence of the C642Kp FMDV gene cassette is shown in FIGS. 20A-20E.

To generate an ALVAC recombinant, primary CEFs were transfected with 20μg of SapI-linearized donor plasmid pCXL-1095-1, using FuGENE-6® reagent(Roche). The transfected cells were subsequently infected with ALVAC asrescue virus at an MOI of 10 and after 29 hours, thetransfected-infected cells were harvested, sonicated, and used forrecombinant virus screening. Recombinant plaques were screened based onthe plaque lift hybridization method using the 1.7 kb FMDV-specificprobe labeled with horseradish peroxidase (HRP) according to themanufacturer's protocol (Amersham). After four sequential rounds ofplaque purification, the recombinant designated as vCP2186.6.2.1.1 wasgenerated and confirmed by hybridization as 100% positive for the FMDVinsert and 100% negative for the C6 ORF.

Single plaques were selected from the 4^(th) round of plaquepurification, and expanded to obtain P1 (1×T25 flask), P2 (1×T75 flask)and P3 (8×roller bottles) amplified stocks. The infected cells from theroller bottles were harvested and concentrated to produce virus stock.The virl concentrate was characterized by performing hybridization ofplaque lifts with the FMDV- and C6-specific probes to confirm 100%genetic purity. Viral DNA was extracted and Southern blotting andsequence analyses confirmed the correct insertion of the FMDV genecassette.

For expression analysis, CEFs were infected at an MOI of 10 with vCP2181(ALVAC C6 I3Lp FMDV P1+3C; see Example 6) or vCP2186 (ALVAC C6 42Kp FMDVP1+3C) and grown at 37° C., in the presence of 5% CO₂, for 24 hours. Thesupernatant was harvested and clarified and the cell monolayer wasresuspended in PBS, and then pelleted. The pellets were resuspended inwater, and then SDS PAGE sample buffer was added to the supernatants.The protein samples were separated on a 10% SDS PAGE gel, thenelectrotransferred to a nylon membrane. The membrane was blocked, andthen probed with rabbit anti-FMDV VP1, VP2, and VP3 antisera. Secondaryantibody and colorimetric analysis revealed that both recombinantsexpressed specific proteins of sizes consistent with VP0, VP1 and VP3 inboth the pellets and supernatants. These data are illustrated in FIG.30.

Example 8 Construction of a pC6 7.5Kp FMDV P1+3C Donor Plasmid for theIntroduction of the FMDV Genes into the Unique C6 Locus of ALVAC

The early 7.5K promoter (7.5Kp) of vaccinia virus (Davison, A. J. andMoss, B. (1989) J. Mol. Biol. 210: 749-769) has been used previously inavipox recombinants.

Plasmid pHM-1119-1 (pUC FMDV, see Example 1) was used as the templatefor PCr amplification of the 7.5K promoter and FMDV genes, up to theunique NdeI site. Primers 11357.CXL and 11358.CXL were used to amplify a1214 bp 7.5Kp 5′-FMDV fragment, which was cloned into pCR2.1, generatingpHM-1249-5-3. The PCR amplification primers are describe in FIG. 21A.

Sequence analysis revealed that three base pair deletions inpHM-1249-5-3. Oligonucleotide primers 11429.HM and 11430.HM weredesigned to re-introduce the missing ucleotides by site-directedmutagenesis. The mutagenesis primers are described I FIG. 21B. Theresultant clones contained 2 of the 3 re-introduced nucleotides, soclone pHM-1267-4 was subjected to a further round of site-directedmutagenesis with primers 11445.HM and 11446.HM. The mutagenesis primersare described in FIG. 21C. Clone pHM-1299-2 (pCR2.1 7.5Kp 5′-FMDV) wasconfirmed to be correct by sequence analysis.

The ˜1.2 kb EcoRI-NdeI fragment from pHM-1299-2 was inserted intopCXL-1013-2 (pC6 FMDV, see Example 3), generating plasmid pHM-1310-4(pC6 7.5Kp FMDV P1+3C). The construction of pHM-1310-4 is illustrated inFIGS. 22A and 22B and the sequence of the C6 7.5Kp FMDV gene cassette isshown in FIGS. 23A-23E.

ALVAC recombinant vCP2189 was obtained after two rounds of screening,but could not be purified/amplified and was lost, suggesting that it wasunstable and/or toxic.

Example 9 Construction of a pC6 Pip FMDV P1+3C Donor Plasmid for theInsertion of the FMDV Genes into the Unique C6 Locus of ALVAC

The early Pi promoter (Pip) from vaccinia virus (Wachsman, M. et al,(1987) J. Infect. Dis. 155: 1188-1197) has been used previously inavipox recombinants. It is 81 nucleotides in length and is thought to bea relatively weak promoter.

Plasmid pHM-1119-1 (pUC FMDV, see Example 1) was used as a template toPCR-amplify the Pip 5′-FMDV fragment, with primers 11356.CXL and11358.CXL (FIG. 24A). The amplified fragment was cloned into pCR2.1 andseveral clones were screened by sequence analysis. The clone with thefewest PCR errors (pHM-1249-4-4, pCR2.1 Pip* 5′-FMDV) was missing 28nucleotides randomly throughout the Pi promoter region, including theEcoRI cloning site.

Oligonucleotides 11395.CXL and 11399.CXL (FIG. 24B) were used toassemble the correct Pi promoter. The Pip was PCR amplified with primers11400.CXL and 11401.CXL (FIG. 24C) and cloned into pCR2.1 to generatepHM-1263-1 (pCR2.1 Pip). Plasmid pHM-1263-1 was used as a template toPCR-amplify a 97 bp Pip 5′-FMDV fragment, using primers 11402.CXL and1140.CXL (FIG. 24D). This fragment contains the EcoRI cloning site, thefull-length Pip and 10 bp of FMDV.

Using pHM-1249-4-4 as template, a 648 bp fragment was PCR-amplifiedusing primers 11406.CXL and 11407.CXL (FIG. 24E). This fragment contains10 bp of the 3′ end of Pip and the 5′-FMDV genes up to a unique KpnIsite.

Equimolar amounts of the 97 bp Pip 5′-FMDV and 648 bp 3′-Pip 5′-FMDV PCRfragments were mixed and amplified using primers 11402.CXL and11407.CXL. The resulting 745 bp Pip 5′-FMDV (EcoRI-KpnI) fragment wascloned into pCR2.1 to generate pHM-1268-1 (pCR2.1 Pip 5′-FMDV,EcoRI-KpnI). The EcoRI-KpnI fragment from pHM-1268-1 was inserted intopHM-1119-1, generating pHM-1277-6 (pUC Pip FMDV).

The 1252 bp EcoRI-NdeI Pip 5′-FMDV fragment from pHM-1277-6 was insertedinto plasmid pCXL-1013-2 (pC6 FMDV P1+3C, see Example 3), generatingplasmid pHM-1284-25 (pC6 Pip FMDV P1+3C). The construction of pC6 PipFMDV P1+3C is illustrated in FIG. 25 and the sequence of the C6 Pip FMDVgene cassette is shown in FIGS. 26A-26E.

ALVAC recombinant vCP2184 was obtained after two rounds of purification,but was lost at the third round of screening/amplification, suggestingthat it was toxic and/or unstable.

Example 10 Construction of a pF8 H6p* FMDV P1+3C Donor Plasmid forInsertion of the FMDV Gene Cassette at the Unique F8 Locus of Fowlpox

Plasmid pHM-1260-2 (pCR2.1 H6p* 5′-FMDV (Nde); see Example 5) was usedas the template to PCR amplify an H6p* 5′-FMDV fragment, with primers11506.HM and 11279.5L. Primer 11506.HM was designed to introduce a HindIII site in front of H6p* and primer 11279.5L was designed to amplifythe FMDV genes up to the unique KpnI site in the VP2 gene. The ˜700 bpfragment was cloned into pCR2.1, generating pHM-1341-7 (pCR2.1 H6p*5′-FMDV KpnI), which was confirmed as correct by sequence analysis. FIG.27 describes the PCR primers.

Plasmid pHM-1180-11 is pF8 H6p FMDV P1+3C, containing the wild-type H6promoter (see Example 2 and FIGS. 5A and 5B). Plasmid pSL-6427-1-1 (pF8MCS) is a promoter-less plasmid used for insertion into the fowlpox F8site. The 0.7 kb HindIII-KpnI H6p* 5′-FMDV fragment from pHM-1341-7 andthe 2.7 kb KpnI-BamH I 3′-FMDV fragment from pHM-1180-11 were ligatedinto plasmid pSL-6427-1-1 that had been digested with HindIII and BamHI,generating pHM-1354-1 (pF8 H6p* FMDV P1+3C). The constructionofpHM-1354-1 is illustrated in FIGS. 28A and 28B and the sequence of theF8 H6p* FMDV P1+3C gene cassette is shown in FIGS. 29A-29F.

To generate a fowlpox recombinant, primary CEFs were transfected withNotI-linearized pHM-1354-1, in the presence of Fugene-6® reagent(Roche). The transfected cells were subsequently infected with fowlpoxas rescue virus at MOI of 10 and after 51 hours, thetransfected-infected cells were harvested, sonicated and used forrecombinant virus screening. Recombinant plaques were screened based onthe plaque lift hybridization method using a 1.7 kb FMDV-specific probelabelled with horseradish peroxidase (HRP) according to themanufacturer's protocol (Amersham Cat# RPN3001). After five sequentialrounds of plaque purification, a fowlpox recombinant designated asvFP2215.1.3.1.1.1 was generated and confirmed by hybridization as 100%positive for the FMDV insert and 100% negative for the F8 ORF.

Single plaques were selected from the 5^(th) round of plaquepurification, and expanded to obtain P1 (1×T25 flask), P2 (2×T75 flask)and P3 (10×roller bottles) stocks to amplify vFP2215. The infected cellsfrom the roller bottles was harvested and concentrated to produce virusstock. The viral concentrate was re-confirmed by hybridization of plaquelifts with the FMDV- and F8-specific probes. Viral DNA was prepared andthe correct insertion of the FMDV gene cassette at the fowlpox F8 locuswas confirmed by Southern blot and sequence analyses.

Example 11 Preparation and Purification of Plasmids

For the preparation of the plasmids intended for the vaccination ofanimals, any technique may be used which makes it possible to obtain asuspension of purified plasmids predominantly in a supercoiled form.These techniques are well known to persons skilled in the art. There maybe mentioned in particular the alkaline lysis technique followed by twosuccessive ultracentrifugations on a caesium chloride gradient in thepresence of ethidium bromide as described in J. Sambrook et al.(Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989). Reference may also be madeto Patent Applications PCT WO 95/21250 and PCT WO 96/02658, whichdescribe methods for producing, on an industrial scale, plasmids whichcan be used for vaccination. For the purposes of the manufacture ofvaccines (see Example 11), the purified plasmids are resuspended so asto obtain solutions at a high concentration (>2 mg/ml), which arecompatible with storage. To do this, the plasmids are resuspended eitherin ultrapure water or in TE buffer (10 mM Tris-HC 1; 1 mM EDTA, pH 8.0).

Example 12 Manufacture of the Associated Vaccine

The various plasmids necessary for the manufacture of an associatedvaccine are mixed starting with their concentrated solutions (Example10). The mixtures are prepared such that the final concentration of eachplasmid corresponds to the effective dose of each plasmid. Thesolutions, which can be used to adjust the final concentration of thevaccine may be either a 0.9M NaCl solution, or PBS buffer.

Specific formulations such as liposomes or cationic lipids may also beused for the manufacture of the vaccines.

Example 13 Vaccination of Animals

The animals are vaccinated with doses of 100 pg, 250 μg or 500 μg perplasmid. The injections are performed with a needle by the intramuscularroute either at the level of the gluteus muscle, or at the level of theneck muscles. The vaccinal doses are administered in volumes of between1 and 5 ml.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

1-30. (canceled)
 31. A method of producing a recombinant avipox vectorcomprising at least one nucleic acid molecule encoding one or morefoot-and-mouth disease virus (FMDV) antigen(s), comprising the steps of:(a) linearizing a donor plasmid with a restriction endonuclease, whereinthe donor plasmid comprises restriction endonuclease cleavage sites or amultiple cloning site; and (b) ligating at least one nucleic acidmolecule comprising (i) a nucleic acid sequence encoding one or moreFMDV antigen(s), (ii) a viral promoter sequence, and (iii) insertionsequences flanking (i) and (ii) that have complementary restrictionendonuclease cleavage sites to the donor plasmid at FMDV antigens,thereby producing the recombinant avipox vector.
 32. The method of claim31, further comprising the steps of: (c) introducing the vector into acell permissive for replication of the vector; and (d) isolating thevector from the cell.
 33. The method of claim 31, wherein the avipox isALVAC.
 34. The method of claim 31, wherein the avipox is fowlpox. 35.The method of claim 31, wherein the antigen comprises at least one ofFMDV VP1, VP2, VP3, VP4, 2A, 2B, and 3C.
 36. The method of claim 31,wherein the nucleic acid sequence encoding one or more FMDV antigen(s)is a cDNA encoding FMDV P1 region and a cDNA encoding FMDV 3C protease.37. The method of claim 31, wherein the promoter sequence is selectedfrom the group consisting of H6 vaccinia promoter, I3L vacciniapromoter, 42K poxyiral promoter, 7.5K vaccinia promoter and Pi vacciniapromoter.
 38. The method of claim 37, wherein the promoter is the H6vaccinia promoter, which is mutated such that expression levels of theFMDV antigens are decreased compared with expression levels of the FMDVantigens under a wild type H6 vaccinia promoter.
 39. The method of claim31, wherein the vector comprises a C6 insertion locus, and whereinflanking sequences of the C6 insertion locus promote homologousrecombination of the FMDV antigens with the C6 insertion locus.
 40. Themethod of claim 39, wherein the flanking sequences comprise C6L and C6Ropen reading frames of avipox.
 41. The method of claim 31, wherein thevector comprises a F8 insertion locus, and wherein flanking sequences ofthe F8 insertion locus promote homologous recombination of the FMDVantigens with the F8 insertion locus.
 42. The method of claim 41,wherein the flanking sequences comprise F8L and F8R open reading framesof avipox.
 43. The method of claim 31, wherein the vector furthercomprises a reporter gene.
 44. The method of claim 43, wherein thereporter gene is selected from the group consisting of neomycinresistance gene, ampicillin resistance gene, lacZ (□-galactosidase),luciferase, and green fluorescent protein (GFP).
 45. The method of claim32, wherein the cell permissive for growth of the vector is a chickenembryonic fibroblast.